WO2011070444A2 - Compositions et procédés pour traiter l'ataxie de friedreich - Google Patents

Compositions et procédés pour traiter l'ataxie de friedreich Download PDF

Info

Publication number
WO2011070444A2
WO2011070444A2 PCT/IB2010/003438 IB2010003438W WO2011070444A2 WO 2011070444 A2 WO2011070444 A2 WO 2011070444A2 IB 2010003438 W IB2010003438 W IB 2010003438W WO 2011070444 A2 WO2011070444 A2 WO 2011070444A2
Authority
WO
WIPO (PCT)
Prior art keywords
frataxin
alkyl
independently
halo
compound
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/IB2010/003438
Other languages
English (en)
Other versions
WO2011070444A3 (fr
Inventor
Roberto Testi
Ottaviano Incani
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Priority to US13/514,288 priority Critical patent/US8703749B2/en
Publication of WO2011070444A2 publication Critical patent/WO2011070444A2/fr
Publication of WO2011070444A3 publication Critical patent/WO2011070444A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/63—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C311/00—Amides of sulfonic acids, i.e. compounds having singly-bound oxygen atoms of sulfo groups replaced by nitrogen atoms, not being part of nitro or nitroso groups
    • C07C311/15—Sulfonamides having sulfur atoms of sulfonamide groups bound to carbon atoms of six-membered aromatic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • the present invention relates generally to compositions and methods useful for the treatment of Friedreich's Ataxia.
  • FRDA Friedreich's Ataxia
  • Dysarthria occurs early in the disease and progress to complete speech impairment. Dysphagia is a late feature and may require artificial feeding. Ventricular hypertrophy characterizes the cardiac picture, and may progressively lead to congestive heart failure and fatal arrhythmias. A significant minority of patients also develop diabetes mellitus, by not yet clearly defined mechanisms 2 .
  • FRDA is caused by homozygous hyperexpansion of GAA triplets within the first intron of the FXN gene, an highly conserved five-exon gene located on the long arm of human chromosome 9, coding for the protein frataxin.
  • Pathological GAA expansions result in "sticky" DNA structures and epigenetic changes that severely reduce transcription of the FXN gene.
  • FRDA patients live with 10-30% residual frataxin, the severity of the disease being directly proportional to the number of GAA triplets and to the consequent degree of frataxin reduction.
  • a minority of FRDA patients, so called compound heterozygotes has pathological GAA expansions on one FXN allele and loss-of-function mutations on the other. Complete loss of frataxin is not compatible with life, in all higher species examined 3 .
  • Human frataxin is synthesized as a 210 amino acid (aa) precursor that is rapidly targeted to the mitochondria. Upon entrance into the mitochondria, the frataxin precursor undergoes a two-step proteolytic processing, mediated by the mitochondrial protein peptidase (MPP). The resulting mature frataxin is a 130aa globular polypeptide that mostly resides within the mitochondrial matrix 4 ' 5 , but that can be also found outside the mitochondria 6 ' 7 , where it might interact with and regulate cytosolic aconitase/IRPl 8 .
  • MPP mitochondrial protein peptidase
  • Frataxin may bind iron directly and act either as an iron donor 9 ' 10 or as an iron sensor involved in the proper functioning of the iron-sulphur cluster (ISC) machinery u . Frataxin-defective cells have reduced activity of ISC-containing enzymes, a general imbalance in intracellular iron distribution and increased sensitivity to oxidative stress.
  • ISC iron-sulphur cluster
  • a first aspect provides a method of treating Friedrich's Ataxia, comprising administering to a subject a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:
  • L is a linking group selected from the group consisting of -S(0) 2 -NH-(CR 2 )x-,
  • R is H or C1-C4 alkyl, and x and y are each independently 0, 1 or 2;
  • each R a and R b is independently Q-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Q-C6 alkoxy, halo, oxo, -NO2, -CF 3 , -CN, -OR9, -SR 9 , -C(0)R 9 , -NHC(0)R 9 , -C(0)OR 9 , -OC(0)R 9 , -NR10R11, -C(O)NRi 0 Rn, -NHR 9 C(O)NRi 0 Rn, or -SO2NR1 0 R11, aryl, arylalkyl, cycloalkyl, or heterocycle, wherein:
  • R 9 , Rio, and Rn are independently H, Q-C6 alkyl, C2-C6 alkenyl, C2-C6
  • alkynyl C1-C6 alkoxy, halo, aryl, arylalkyl, cycloalkyl, or heterocycle, each being optionally substituted with one to four substituents, and two R a or two R b together with the atoms to which they attach on the ring optionally form a ring; s is 0, 1 , 2 or 3; and t is 1 , 2, 3 or 4.
  • each R a is independently Q-C6 alkyl, halo, -NO2, -CF 3 , -CN, or -OR 9 .
  • each R is independently Q-C6 alkyl, halo, -C(0)OR 9 , -C(0)R 9 , -N0 2 , -CF 3 , -CN, or -OR 9 .
  • B is selected from the group consisting of a phenyl group, an imidazole, a pyridine and a pyrimidine.
  • the compound is of formula la:
  • the compound is of formula lb:
  • the compound has the structure of formula Id:
  • R is halo, Ci-Ce alkyl, C1-C6 alkoxy, or -NO2; R 4 and R 8 are independently H, -OH, or Q-C6 alkoxy; and R 5 and R 7 are independently H, halo, or -NO2.
  • At least one R b is -N0 2 .
  • t is 2 or 3 and at least one R b is a halog and at least one R b is -N0 2 .
  • s is 1 , 2 or 3 and at least one R a is halogen.
  • the compound has the structure of formula XII:
  • the method of treating Friedreich's Ataxia comprises inhibiting ubiquitination of frataxin.
  • the method of treating Friedreich's Ataxia comprises elevating intracellular frataxin levels.
  • a second aspect provides a method of elevating intracellular frataxin levels.
  • elevating intracellular frataxin levels is accomplished by inhibiting ubiquitination of frataxin.
  • elevating intracellular frataxin levels is accomplished by blocking binding by ubiquitin of lysine 147 of frataxin.
  • the blocking of binding by ubiquitin of lysine 147 of frataxin is accomplished by a compound having the structure of formula I, la, lb, Ic, Id or XII.
  • a third aspect provides a method of inhibiting the ubiquitination of frataxin, comprising blocking binding by ubiquitin of lysine 147 of frataxin, wherein frataxin has the sequence of SEQ ID O: l .
  • the blocking of binding by ubiquitin of lysine 147 of frataxin is accomplished by a compound having the structure of formula I, la, lb, Ic, Id or XII.
  • a fourth aspect provides a method of identifying an agent that inhibits ubiquitination of frataxin thereby elevating the intracellular levels of frataxin, comprising the steps of: providing an agent; contacting the agent with frataxin or a fragment thereof;
  • determining whether said agent inhibits ubiquitination of frataxin comprises measuring intracellular frataxin levels.
  • the method further comprises the step of designing an agent based upon a three-dimensional structure of a frataxin binding pocket defined by the structural coordinates of at least amino acid residues 92-106, 126-132, and 144-156 of native frataxin (SEQ ID: 1).
  • the three-dimensional structure of the frataxin binding pocket is determined using X-ray crystallography.
  • the three-dimensional structure of the frataxin binding pocket is determined using protein NMR.
  • compositions comprising a compound of one of structures I, la, lb, Ic, Id or XII are provided.
  • Fig. 1 illustrates that frataxin levels are controlled by proteasome-mediated degradataion.
  • Fig. 2 illustrates that frataxin can be mono- and poly-ubiquitinated in vivo.
  • Fig. 3 illustrates that K 147 is the main ubiquitination target.
  • Fig. 4 illustrates that K 147 is part of a druggable molecular surface.
  • Fig. 5 illustrates that ubiquitin-competing molecules prevent frataxin
  • Fig. 6 illustrates that ubiquitin-competing molecules induce frataxin accumulation and rescue both aconitase and ATP defects in FRDA lymphoblasts.
  • Fig. 7 illustrates that ubiquitin-competing molecules induce frataxin accumulation and rescue ATP defects in FRDA fibroblasts.
  • Fig. 8 illustrates that ubiquitin-competing molecules induce frataxin accumulation in HEK-293 cells.
  • Fig. 9 illustrates additional compounds are effective in inducing frataxin accumulation in FRDA lymphoblasts.
  • compositions and methods for the treatment of Freidrich's Ataxia are compositions and methods for the treatment of Freidrich's Ataxia.
  • the present disclosure relates to the surprising discovery that Freidrich's Ataxia can be treated by inhibiting degradation of frataxin by the Ubiquitin-Proteasome System. Frataxin is directly modified by ubiquitin, and lysine 147 is the critical residue responsible for frataxin ubiquitination and subsequent degradation.
  • Described herein are compounds and methods for treating Friedreich's Ataxia.
  • methods of treating Friedreich's Ataxia are described, wherein the frataxin molecular pocket harboring lysine 147 is targeted.
  • methods for inhibiting frataxin ubiquitination and degradation are described, wherein the molecular pocket harboring lysine 147 is targeted.
  • methods for increasing frataxin levels are described, wherein the molecular pocket harboring lysine 147 is targeted.
  • the present disclosure provides a description of the molecular pocket harboring lysine 147 .
  • methods of blocking ubiquitin from accessing the molecular pocket harboring lysine 147 are provided.
  • compounds are provided for inhibiting ubiquitin-mediated degradation by targeting the frataxin molecular pocket harboring lysine 147 .
  • the compounds of the present disclosure may be any compound capable of inhibiting ubiquitin-mediated degradation of frataxin by targeting the molecular pocket harboring lysine 147 .
  • the compounds of the present disclosure may be small molecules, peptides, or any agent capable of targeting the molecular pocket.
  • the compounds of the present disclosure are used to treat Friedreich's Ataxia by binding and blocking the frataxin molecular pocket harboring lysine 147 . In further aspects, the compounds of the present disclosure are used to increase frataxin levels by binding and blocking the frataxin molecular pocket harboring lysine 147 .
  • alkenyl as used herein contemplates substituted or unsubstituted, straight and branched chain alkene radicals, including both the E- and Z-forms, containing from two to eight carbon atoms.
  • the alkenyl group may be optionally substituted with one or more substituents selected from the group consisting of Q-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Q-C6 alkoxy, halo, aryl, arylalkyl, cycloalkyl, or heterocycle.
  • alkyl as used herein contemplates substituted or unsubstituted, straight and branched chain alkyl radicals containing from one to fifteen carbon atoms.
  • the alkyl group may be optionally substituted with one or more substituents selected from Q-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Q-C6 alkoxy, halo, aryl, arylalkyl, cycloalkyl, or heterocycle.
  • alkoxy contemplates an oxygen with a Ci-Ce alkyl group as a substituent and includes methoxy, ethoxy, butoxy, trifluromethoxy and the like. It also includes divalent substituents linked to two separated oxygen atoms such as, without limitation, -0-(CH 2 )i- 4 -0-, -O-CF2-O-, -0-(CH 2 )i- 4 -0-(CH 2 CH 2 -0)i- 4 - and
  • alkynyl as used herein contemplates substituted or unsubstituted, straight and branched carbon chain containing from two to eight carbon atoms and having at least one carbon-carbon triple bond.
  • alkynyl includes, for example ethynyl, 1-propynyl, 2- propynyl, 1-butynyl, 3 -methyl- 1-butynyl and the like.
  • the alkynyl group may be optionally substituted with one or more substituents selected from Q-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Q-C6 alkoxy, halo, aryl, arylalkyl, cycloalkyl, or heterocycle.
  • Antibody refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • the antigen-binding region of an antibody will be most critical in specificity and affinity of binding.
  • aryl as used herein contemplates substituted or unsubstituted single- ring and multiple aromatic groups (for example, phenyl, pyridyl and pyrazole, etc.) and polycyclic ring systems (naphthyl and quinolinyl, etc.).
  • the polycyclic rings may have two or more rings in which two atoms are common to two adjoining rings (the rings are "fused") wherein at least one of the rings is aromatic, e.g., the other rings can be cycloalkyls, cycloalkenyls, aryl, heterocycles and/or heteroaryls.
  • the aryl group may be optionally substituted with one or more substituents selected from Q-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Q-C6 alkoxy, halo, aryl, arylalkyl, cycloalkyl, or heterocycle.
  • arylalkyl as used herein contemplates a Ci-Ce alkyl group which has as a substituent an aromatic group, which aromatic group may be substituted or unsubstituted.
  • the aralkyl group may be optionally substituted with one or more substituents selected from C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Q-C6 alkoxy, halo, aryl, arylalkyl, cycloalkyl, or heterocycle.
  • cycloalkyl as used herein contemplates substituted or unsubstituted cyclic alkyl radicals containing from three to twelve carbon atoms and includes cyclopropyl, cyclopentyl, cyclohexyl and the like.
  • cycloalkyl also includes polycyclic systems having two rings in which two or more atoms are common to two adjoining rings (the rings are "fused").
  • the cycloalkyl group may be optionally substituted with one or more substituents selected from Q-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Q-C6 alkoxy, halo, aryl, arylalkyl, cycloalkyl, or heterocycle.
  • halo or halogen as used herein includes fluorine, chlorine, bromine and iodine.
  • heterocycle contemplates substituted or unsubstituted aromatic and non-aromatic cyclic radicals having at least one heteroatom as a ring member.
  • Preferred heterocyclic groups are those containing five or six ring atoms which includes at least one hetero atom and includes cyclic amines such as morpholino, piperidino, pyrrolidino and the like and cyclic ethers, such as tetrahydrofuran, tetrahydropyran and the like.
  • Aromatic heterocyclic groups also termed "heteroaryl” groups, contemplates single-ring hetero-aromatic groups that may include from one to three heteroatoms, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, oxodiazole, thiadiazole, pyridine, pyrazine, pyridazine, pyrimidine and the like.
  • heteroaryl also includes polycyclic hetero-aromatic systems having two or more rings in which two or more atoms are common to two adjoining rings (the rings are "fused") wherein at least one of the rings is a heteroaryl, e.g., the other rings can be cycloalkyls, cycloalkenyls, aryl, heterocycles and/or heteroaryls.
  • polycyclic heteroaromatic systems examples include quinoline, isoquinoline, cinnoline, tetrahydroisoquinoline, quinoxaline, quinazoline, benzimidazole, benzofuran, benzothiophene, benzoxazole, benzothiazole, indazole, purine, benzotriazole,
  • heterocyclic group may be optionally substituted with one or more substituents selected from the group consisting of Q-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Q-C6 alkoxy, halo, aryl, arylalkyl, cycloalkyl, or heterocycle.
  • “Pharmaceutically acceptable salt” refers to a salt of a compound of the invention which is made with counterions understood in the art to be generally acceptable for pharmaceutical uses and which possesses the desired pharmacological activity of the parent compound.
  • Such salts include: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid,
  • cyclopentanepropionic acid glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid,
  • salts of amino acids such as arginates and the like, and salts of organic acids like glucurmic or galactunoric acids and the like (see, e.g., Berge et al, 1911 , J. Pharm. Set 66: 1-19).
  • a first aspect provides a method of treating Friedrich's Ataxia, comprising administering to a subject a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:
  • L is a linking group selected from the group consisting of -S(0) 2 -NH-(CR 2 ) x -,
  • R is H or C1-C4 alkyl, and x and y are each independently 0, 1 or 2;
  • each R a and R b is independently Q-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, Q-C 6 alkoxy, halo, oxo, -N0 2 , -CF 3 , -CN, -OR9, -SR 9 , -C(0)R 9 , -NHC(0)R 9 , - C(0)OR 9 , -OC(0)R 9 , -NR10R11, -C(O)NRi 0 Rn, -NHR 9 C(O)NRi 0 Rn, or - S0 2 NRioRii, aryl, arylalkyl, cycloalkyl, or heterocycle, wherein:
  • R 9 , Rio, and Rn are independently H, -C6 alkyl, C 2 -C6 alkenyl, C 2 -C6
  • alkynyl C1-C6 alkoxy, halo, aryl, arylalkyl, cycloalkyl, or heterocycle, each being optionally substituted with one to four substituents, and two R a or two R b together with the atoms to which they attach on the ring optionally form a ring; s is 0, 1, 2 or 3; and t is 1, 2, 3 or 4.
  • each R a is independently Q-C6 alkyl, halo, -NO2, -CF 3 , -CN, or -OR 9 .
  • each R b is independently Q-C6 alkyl, halo, -C(0)OR 9 , -C(0)R 9 , -N0 2 , -CF 3 , -CN, or -OR 9 .
  • B is selected from the group consisting of a phenyl group, an imidazole, a pyridine and a pyrimidine.
  • the compound is of formula la:
  • the compound is of formula Ic:
  • the compound has the structure of formula Id: wherein R a is halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, or - O2; R 4 and R 8 are independently H, -OH, or Q-C6 alkoxy; and R 5 and R 7 are independently H, halo, or -NO2.
  • At least one R b is -N0 2 .
  • t is 2 or 3 and at least one R b is a halog and at least one R b is -N0 2 .
  • s is 1, 2 or 3 and at least one R a is halogen.
  • the compound has the structure of formula XII:
  • the compound has the structure of
  • Example compounds to be used with the disclosed methods have been identified through the screening methods disclosed herein. These compounds include: IV(aka SC620301); V(aka SC620304);
  • a compound useful in the disclosed method of formula I is:
  • the method of treating Friedrich's Ataxia comprises inhibiting ubiquitination of frataxin.
  • the method of treating Friedreich's Ataxia comprises elevating intracellular frataxin levels.
  • a second aspect provides a method of elevating intracellular frataxin levels.
  • elevating intracellular frataxin levels is accomplished by inhibiting ubiquitination of frataxin.
  • elevating intracellular frataxin levels is accomplished by blocking binding by ubiquitin at lysine 147 of frataxin.
  • the blocking of binding by ubiquitin of lysine 147 of frataxin is accomplished by any of the compounds of any of the formulae disclosed with respect to the first aspect
  • a third aspect provides a method of inhibiting the ubiquitination of frataxin, comprising blocking binding by ubiquitin of lysine 147 of frataxin, wherein frataxin has the sequence of SEQ ID O: l .
  • the blocking of binding by ubiquitin of lysine 147 of frataxin is accomplished by any of the compounds of any of the formulae disclosed with respect to the first aspect.
  • the compounds disclosed with respect to the first aspect inhibit ubiquitin-mediated degradation by binding and blocking the frataxin molecular pocket harboring lysine 147 .
  • these compounds are used to treat Friedreich's Ataxia by binding and blocking the frataxin molecular pocket harboring lysine 147 .
  • these compounds are used to increase frataxin levels by binding and blocking the frataxin molecular pocket harboring lysine 147 .
  • a fourth aspect provides a method of identifying an agent that inhibits ubiquitination of frataxin, comprising the steps of: providing an agent; contacting the agent with frataxin or a fragment thereof; measuring a signal correlated with a lack of
  • determining whether said agent inhibits ubiquitination of frataxin comprises measuring intracellular frataxin levels.
  • the method further comprises the step of designing an agent based upon a three-dimensional structure of a frataxin binding pocket defined by the structural coordinates of at least amino acid residues 92-106, 126-132, and 144-156 of native frataxin (SEQ ID: 1).
  • the three-dimensional structure of the frataxin binding pocket is determined using X-ray crystallography.
  • the three-dimensional structure of the frataxin binding pocket is determined using protein NMR.
  • compositions comprising a compound of one of structures of any one of the formulae disclosed with respect to the first aspect is provided.
  • the compounds described herein can be used as pharmaceutical compositions comprising the compounds, together with one or more pharmaceutically acceptable excipients or vehicles, and optionally other therapeutic and/or prophylactic ingredients.
  • excipients include liquids such as water, saline, glycerol, polyethyleneglycol, hyaluronic acid, ethanol, cyclodextrins, modified cyclodextrins (i.e., sufobutyl ether cyclodextrins) etc.
  • Suitable excipients for non-liquid formulations are also known to those of skill in the art.
  • Pharmaceutically acceptable salts can be used in the compositions of the present invention and include, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like
  • organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • auxiliary substances such as wetting or emulsifying agents, biological buffering substances, surfactants, and the like, may be present in such vehicles.
  • a biological buffer can be virtually any solution which is pharmacologically acceptable and which provides the formulation with the desired pH, i.e., a pH in the physiologically acceptable range. Examples of buffer solutions include saline, phosphate buffered saline, Tris buffered saline, Hank's buffered saline, and the like.
  • the pharmaceutical compositions may be in the form of solid, semi-solid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, suspensions, creams, ointments, lotions or the like, preferably in unit dosage form suitable for single administration of a precise dosage.
  • the compositions will include an effective amount of the selected drug in combination with a pharmaceutically acceptable carrier and, in addition, may include other pharmaceutical agents, adjuvants, diluents, buffers, etc.
  • the invention includes a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of the present invention including isomers, tautomers, racemic or non-racemic mixtures of isomers, or pharmaceutically acceptable salts or solvates thereof together with one or more pharmaceutically acceptable carriers, and optionally other therapeutic and/or prophylactic ingredients.
  • conventional nontoxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, etc., an active compound as described herein and optional pharmaceutical adjuvants in an excipient, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension.
  • the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, tonicifying agents, and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate,
  • the composition will generally take the form of a tablet, capsule, a softgel capsule or may be an aqueous or nonaqueous solution, suspension or syrup. Tablets and capsules are preferred oral administration forms. Tablets and capsules for oral use will generally include one or more commonly used carriers such as lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. When liquid suspensions are used, the active agent may be combined with emulsifying and suspending agents. If desired, flavoring, coloring and/or sweetening agents may be added as well. Other optional components for incorporation into an oral formulation herein include, but are not limited to, preservatives, suspending agents, thickening agents, and the like.
  • a pharmaceutically or therapeutically effective amount of the composition will be delivered to the subject.
  • the precise effective amount will vary from subject to subject and will depend upon the species, age, the subject's size and health, the nature and extent of the condition being treated, recommendations of the treating physician, and the therapeutics or combination of therapeutics selected for administration. Thus, the effective amount for a given situation can be determined by routine experimentation.
  • the subject may be administered as many doses as is required to reduce and/or alleviate the signs, symptoms, or causes of the disorder in question, or bring about any other desired alteration of a biological system.
  • Frataxin Binding Site Analysis The crystal structure of human frataxin 19 20 was employed to characterize regions of buried volume and to identify positions likely to represent binding sites based upon the size, shape, and burial extent of these volumes using the program PASS 21 . This analysis identified 8 putative binding sites, one of which, located around Lysl47, was used in the virtual screening and docking studies.
  • Target preparation The virtual screening studies were performed with AutoDock version 4.2 and AutoDock Vina version 1 22 .
  • the subset obtained from cluster analysis (similarity threshold of 60%, "ncid_t60” cluster from ZINC, 9218 compounds), filtered to select lead-like compounds (ZINC subset "lead-like", about 316,000 compounds, ), produced a relatively small dataset of 6,025 structures. These molecules were used in the virtual screening procedure.
  • HEK-293 cells were transiently transfected with frataxin-GFP fusion protein, using the Ca/Ph precipitation method in 10 cm plates. After 18 hrs, cells were splitted, pooled and plated again in 96 wells, to normalize for variation in transfection efficiency. Each molecule was then added at 50 or 100 ⁇ to 12 independent wells. MG132 was used as a positive control. 18 hrs after treatment, cells from all wells were collected and pooled together. Cells were then fixed in 4% paraformaldehyde and changes in fluorescence intensity were monitored by FACS analysis. Small molecules increasing fluorescence intensity, similarly to MG132, were re-tested for their effects on both GFP alone and frataxin-GFP to identify molecules specifically affecting frataxin levels.
  • HEK-293 Human embryonic kidney HEK-293 cells and Hela cells were maintained in DMEM supplemented with 10% FBS. HEK-293 were transfected with the calcium/phosphate precipitation method, using 20 ⁇ g total DNA (10 ⁇ g pIRES-frataxin and 10 ⁇ g HA-Ub, or corresponding empty vectors) on 10 cm dishes. Hela cells were transfected using Lipofectamine 2000 reagents (Invitrogen), according to manufacturer's instructions. Where indicated, the day after transfection, cells were treated for 16 h with 10 ⁇ proteasome inhibitors, MG132 or Lactacystin, or 50 ng/ml DUB inhibitor Ubiquitin-Aldehyde.
  • Flp-In-HEK-293 cells are HEK-293 variants allowing the stable and isogenic integration and expression of a transfected gene.
  • Flp-In-HEK-293 cells were maintained in DMEM supplemented with 10% FBS and transfected with the calcium/phosphate precipitation method. Briefly, cells were plated on 10 cm dishes and transfected with 10 ⁇ g total DNA.
  • the HEK-293 clone stably expressing frataxin 1 210 was previously described (4).
  • the HEK-293 clone stably expressing frataxin K147R was obtained from cultures in selection medium containing 100 ⁇ g/ml hygromycin B (Invitrogen).
  • FRDA lymphoblasts GM15850, GM16798 and GM16241, as well control lymphoblasts GM15851, GM 16241 and GM16215 were maintained in RPMI supplemented with 15% FBS.
  • Treatments with specific ubiquitin-competing molecules were performed in 20% FBS containing medium.
  • FRDA fibroblasts GM03816 were maintained in DMEM supplemented with 15% FBS.
  • Antibodies The following antibodies were used for immunoprecipitation and westrern blot analysis: mAb anti-frataxin (MAB- 10876, Immunological Science), mAb anti- HA (clone HA-7, Sigma), mAb anti-Ubiquitin (clone P4D1, Santa Cruz), mAb anti-tubulin (Sigma), secondary antibody HRP -conjugated goat anti-mouse (Pierce).
  • mAb anti- frataxin MAB- 10485, Immunological Science
  • mAb anti-Bcl2 sc-509, Santa Cruz
  • FITC-conjugated goat anti-mouse IgG/IgM BD Bioscience Pharmingen
  • Proteasome inhibitors MG132 and Lactacystin (Sigma Aldrich); DUB inhibitors: Ub-Aldehyde (Biomol) and N-Ethylmaleimide ( ⁇ , Sigma Aldrich). Protein synthesis inhibitor: Cycloheximide (Sigma Aldrich).
  • DNA constructs The pIRES2-frataxin 1 210 construct was previously described (Condo, I. et al, 2006). All the lysines mutants constructs were generated using the Quick- Change site-directed mutagenesis kit (Stratagene) with specific primers using pIRES2- frataxin 1 210 as template. All the constructs generated were verified by DNA sequencing. The Ha-Ub construct was generated by M. Treier in Dirk Bohmann's lab (Treier, M. et al. 1994). The pEGFP -frataxin construct was generated from pIRES2- frataxin 1"210 by PCR
  • deoxycholate 0, 1% SDS, 1% Np40, 2mM EDTA
  • IP buffer 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 5 mM EDTA, 5 mM EGTA
  • Complete protease inhibitor cocktail and 2 mM N-Ethylmaleimide (NEM).
  • NEM N-Ethylmaleimide
  • Aconitase assay and determination of ATP were washed twice with ice-cold Dulbecco's Phosphate Buffered Saline (DPBS) and lysed in CelLytic M buffer (Sigma-Aldrich) supplemented with Complete protease inhibitor cocktail, EDTA-free (Roche).
  • DPBS Dulbecco's Phosphate Buffered Saline
  • CelLytic M buffer CelLytic M buffer
  • EDTA-free Roche
  • Aconitase activity was measured spectrophotometrically at 340 nm by a coupled reaction of aconitase and isocitrate dehydrogenase.
  • the assay reactions contained 100 mg of cell extract in 50 mM Hepes pH 7.4, 1 mM sodium citrate, 0.6 mM MnCl 2 , 0.2 mM NADP + and 2 U/ml isocitrate dehydrogenase (Sigma-Aldrich). Citrate synthase activity was assessed using 10 mg of cell extract with the Citrate Synthase Assay Kit (Sigma-Aldrich CS0720). The aconitase activities were normalized with respect to citrate synthase ratios; one milliunit of enzyme was defined as the amount of protein that converted 1 nmol of NADP + in 1 min at 25°C.
  • HEK-293 Flp-In cells stably transfected with frataxin 1"210 were treated for the indicated times with 10 ⁇ MG132. Total cell extracts were blotted as in A.
  • One representative experiment out of four performed with similar results is shown in Fig. IB.
  • FIG. 1C and ID show quantitative analysis of frataxin precursor and mature accumulation upon MG132 treatment of HEK-293 Flp-In cells, as shown in IB.
  • HEK-293 Flp-In cells stably transfected with frataxin 1"210 (FIG IE) or empty vector (FIG 1G) were treated for the indicated times with 100 ⁇ g/ml cycloheximide (CHX) in the presence or absence of 10 ⁇ MG132 (MG).
  • CHX cycloheximide
  • MG132 MG132
  • Fig. IF and 1H illustrate densitometric analysis of the expression of frataxin precursor as shown in E and G, respectively, normalized to tubulin levels. Dotted line indicates frataxin precursor half-life.
  • the frataxin 1"210 precursor is rapidly imported in the mitochondrial matrix, where it is quantitatively processed to generate mature frataxin 81"210 4 ' 5 . Since proteins within the mitochondrial matrix are shielded from UPS degradation, whether the UPS could affect the stability of the frataxin precursor was determined. To address this question, the proteasome in HeLa cells, transiently transfected with frataxin 1"210 to allow for sufficient precursor accumulation was inhibited .
  • Fig. 1A shows that cells treated with proteasome inhibitors lactacystin (LC) or MG132 (MG) accumulated significantly higher amounts of precursor compared to untreated cells.
  • HEK-293 Flp-In cells stably expressing frataxin 1"210 were used.
  • This cell line is engineered to integrate a single copy of the transfected cDNA and therefore, unlike transiently transfected cells, it allows the accumulation of frataxin products at more physiologic levels.
  • MG132 a time-dependent and quite remarkable (>15 fold after 24 hrs) accumulation of the frataxin precursor was observed.
  • a ⁇ 2.5 fold accumulation of mature frataxin was also detected after 24 hrs of treatment (Fig. 1B-D).
  • Frataxin can be mono- and poly-ubiquitinated in vivo.
  • Fig. 2A illustrates HEK- 293 cells transiently transfected with frataxin 1"210 and HA-tagged ubiquitin (HA-Ub) (where indicated) were treated with 10 ⁇ MG132 (MG) for 16 h.
  • MG MG132
  • One representative experiment out of five performed with similar results is shown.
  • Total cell extracts (lanes 1-4) or anti-HA immunoprecipitates (lanes 5-8) were analyzed by WB with anti-frataxin antibody.
  • FIG. 2B illustrates HEK-293 cells transiently transfected with frataxin 1"210 and HA-Ub (where indicated) or control empty vector (ev) were treated as above. Polyubiquitin- conjugated forms of frataxin were detected by WB with anti-ubiquitin antibody on immunoprecipitated frataxin. One representative experiment out of three performed with similar results is shown.
  • Protein degradation through the proteasome is a highly specific process that implies as a first step the conjugation of one or more ubiquitin molecules to the protein to be degraded.
  • HEK-293 cells were transiently co-transfected with frataxin 1"210 and HA-tagged ubiquitin (HA-Ub), in the presence of MG132.
  • Fig. 2A shows that, when HA-Ub is co-transfected with frataxin 1"210 , and only in the presence of MG132, bands migrating slower than the precursor are recognized by anti-frataxin mAbs, consistent with the accumulation of mono-ubiquitinated frataxin amid proteasome inhibition (lane 4).
  • K 147 is the main ubiquitination target.
  • HEK-293 cells transiently transfected with HA-tagged ubiquitin (HA-Ub) and frataxin 1"210 or K 147 -mutant frataxin (K147R) were treated with 10 ⁇ MG132 (MG) for 16 h.
  • Anti-HA immunoprecipitates were analyzed by WB with anti-frataxin antibody to detect ubiquitin-conjugated frataxin.
  • Fig. 3A One representative experiment out of five performed with similar results is shown in Fig. 3A.
  • HEK-293 cells transiently transfected with HA-Ub and the lysine-less frataxin mutant (13KR) or the lysine-less frataxin mutant in which K 147 has been reintroduced (13KR- R147K) were treated with 10 ⁇ MG132 for 16 h.
  • Anti-HA immunoprecipitates were analyzed as in A.
  • One representative experiment out of two performed with similar results is shown in Fig. 3B.
  • HEK-293 Flp-In cells stably expressing frataxin 1"210 (HEK-293-frataxin) or the K147R frataxin mutant (HEK-293-frataxin K147R ) were treated for the indicated times with 100 ⁇ g/ml cycloheximide (CHX) to block new protein synthesis. Proteins were resolved on SDS- PAGE and revealed with anti-frataxin antibody or anti-tubulin, as a loading control. Pre: frataxin precursor.
  • Fig. 3C One representative experiment out of three performed with similar results is shown in Fig. 3C.
  • Frataxin is resistant ttoo UUPFS-mediated degradation.
  • the loss of the ubiquitin docking site should grant the frataxin K147R mutant a relative resistance to UPS-mediated degradation, thus increasing its stability.
  • frataxin K147R was stably expressed in HEK-293 cells. After exposure to cycloheximide to block new protein synthesis, the stability of the frataxin 47R precursor was monitored over time and compared to the stability of frataxin precursor of HEK-293 cells stably expressing wild type frataxin 1"210 and similarly treated.
  • Fig. 3C-D shows that the frataxin K147R precursor is significantly more stable (-45% of the input after 24 h) than the frataxin 1"210 precursor (-15% of the input after 24 h).
  • K 147 is part of a druggable molecular surface.
  • Fig. 4A illustrates solvent- accessible surface of frataxin.
  • the binding site near K 147 includes E 96 , E 100 , D 104 , F 127 , G 130 , L 103 , and A 99 .
  • the latter aminoacid, omitted for clarity, is at the left of L 103 at the bottom of the cleft.
  • Fig. 4B is a cartoon representation of frataxin illustrating charged residues of the putative binding surface near K 147 .
  • E 96 is likely to form a stabilizing bond with K 147 .
  • Fig. 4C illustrates the compound Formula III on the molecular surface of frataxin.
  • Fig. 4D illustrates selected interactions between frataxin and the ligand.
  • Ubiquitin-competing molecules prevent frataxin ubiquitination.
  • Formula III prevents frataxin ubiquitination.
  • HEK-293 cells were transiently co-transfected with HA-Ub and either frataxin 1"210 or lysine-mutant frataxin (K147R). Where indicated, cells were pretreated with 20 ⁇ or 50 ⁇ formula III one hour before transfection. The molecule was re-added 24 hours after transfection and cells were harvested 48 hours after transfection. Where indicated, cells were also treated with 10 ⁇ MG132 for the last 16 hours. Total cell extracts (upper panel) or anti-HA immunoprecipitated proteins (lower panel) were detected with anti-frataxin antibody.
  • Fig. 5A One representative experiment out of three performed with similar results is shown in Fig. 5A.
  • Formula III induces frataxin precursor accumulation.
  • HEK-293 Flp-In cells stably expressing frataxin 1"210 were treated for the indicated days with 20 ⁇ of Formula III or 10 ⁇ MG132.
  • Total cell extracts were resolved on SDS-PAGE and analyzed with anti-frataxin antibody, or anti-tubulin, as a loading control.
  • Fig. 5B One representative experiment out of three performed with similar results is shown in Fig. 5B.
  • Formula III induces mature frataxin accumulation.
  • HEK-293 Flp-In cells stably expressing frataxin 1"210 were treated and analyzed as in B.
  • One representative experiment out of three performed with similar results is shown Fig. 5C.
  • HEK-293 cells were transiently co-transfected with HA-tagged ubiquitin (HA-Ub) and frataxin 1"210 , in the presence of 20 ⁇ and 50 ⁇ of Formula III (Fig. 5A). Ubiquitinated frataxin was revealed after 48 h, by Western Blotting of total cell lysates (upper panel) and of anti-HA
  • HEK-293 cells were also transiently co-transfected with HA-tagged ubiquitin (HA-Ub) and the frataxin K147R mutant (K147R) that lacks the ubiquitinable lysine, as a negative control.
  • HA-Ub HA-tagged ubiquitin
  • K147R frataxin K147R mutant
  • Preventing ubiquitination should result in a reduced degradation and consequent accumulation of frataxin.
  • HEK-293 Flp-In cells stably expressing frataxin 1"210 were exposed to Formula III for the days indicated, and the accumulation of the frataxin precursor (Fig. 5B) and mature frataxin (Fig. 5C) was quantitated by WB.
  • the treatment of HEK-293 cells stably expressing frataxin with Formula III is able to induce substantial accumulation of both the frataxin precursor and, over a longer time period, of mature frataxin.
  • Ubiquitin-competing molecules are effective in FRDA cells.
  • FRDA lymphoblasts GM15850 were cultured for 6 days in the presence of 50 ⁇ Formula IV or Formula VI. Cells were then fixed, stained with anti-frataxin antibody or anti-Bcl2, as a control, and analyzed by flow cytometry.
  • Fig. 6A One representative experiment out of three performed with similar results is shown as Fig. 6A.
  • FRDA lymphoblasts GM15850, GM16798 and GM16214 were left untreated or cultured for 6 days in the presence of 50 ⁇ Formula IV. Their respective genetically-related healthy control GM15851, GM 16241 and GM16215 lymphoblasts were left untreated and shown for comparison in Fig. 6B. Total cell extracts were resolved on SDS-PAGE and analyzed with anti-frataxin antibody, or anti-tubulin, as a loading control.
  • FRDA lymphoblasts GM16798 were left untreated or treated for 6 days with 50 ⁇ Formula IV. Their genetically-related healthy control GM 16241 lymphoblasts were left untreated.
  • Fig. 6C shows the results. Aconitase activity and ATP levels were measured as described previously.
  • GM15850 cells Lymphoblasts derived from a FRDA patient were therefore exposed to these compounds for different time periods.
  • FACS analysis reveals a discrete frataxin accumulation detectable in all cells after 6 days of treatment with both molecules.
  • the accumulation of mature frataxin can be detected by SDS-PAGE and western blot analysis in GM15850 cells, as well as in lymphoblasts derived by two additional FRDA patients (GM 16798 and
  • GM16214 cells exposed to Formula IV for 6 days. Frataxin levels in the respective genetically-related healthy control-derived cell lines are also shown for comparison.
  • FIG. 6C shows that exposure of GM16798 lymphoblasts to Formula IV is able to significantly boost both aconitase activity and ATP levels after 6 days of treatment. Aconitase and ATP levels of the respective genetically -related healthy control-derived lymphoblasts are also shown for comparison.
  • FRDA fibroblasts (GM03816 cells) were exposed to compound Formula IV for different time periods Fig. 7 shows that frataxin accumulation can be detected as early as 3 days of treatment by both FACS analysis or SDS-PAGE and western blot analysis. Rescue of ATP levels can also be achieved in GM03816 fibroblasts exposed to Formula IV after 3 days of treatment (Fig. 7).
  • FRDA fibroblasts GM03816 were treated for 3 days with 100 ⁇ Formula IV. Cells were then fixed, stained with anti-frataxin antibody or anti-Bcl2, as a control, and analyzed by flow cytometry.
  • Fig. 7 A One representative experiment out of three performed with similar results is shown in Fig. 7 A.
  • FRDA fibroblasts GM03816 were treated as above. Total cell extracts were resolved on SDS-PAGE and analyzed with anti- frataxin antibody, or anti-tubulin, as a loading control. Fig. 7B shows the results. FRDA fibroblasts GM03816 were treated as above. ATP levels were quantitated as described previously. Results are shown in Fig. 7C.
  • Alinda IVK/1053144 and IVK/107021 1 are effective in inducing mature frataxin accumulation in FRDA cells.
  • FRDA lymphoblasts (GM15850) were treated for 3 days with 100 mM of IVK/1053144 or IVK/1070211 (Alinda codes).
  • Total cell extracts from treated cells and from untreated cells (-) were resolved on SDS-PAGE and analyzed with anti- frataxin antibody, or anti-tubulin antibody.
  • Fig. 9 shows the results.
  • mice that express only the human i3 ⁇ 4N gene in which a GAA expansion has been inserted will be used. These mice therefore lack murine frataxin and produce low levels of human frataxin. They show a variety of neupathological signs that mimic the human disease, thus are currently considered the animal model that more closely represents the human FRDA genetic defect. This will verify that compounds that elevate frataxin in FRDA cells in vitro are also able to elevate frataxin levels in tissues and alleviate the pathology and the clinical picture in the FRDA mice.
  • FRDA mice will be treated at the doses indicated by preliminary toxicity studies in normal mice, and different regiments will be investigated. At the appropriate times, different biochemical, behavioural and histopathological parameters will be investigated. Locomotor activity will be assessed by examining the unrestricted movement of mice.
  • Coordination ability will be quantitated using an accelerating rotarod treadmill. Muscle strength will be measured by a forelimb grip test. Sections of the brain, spinal cord and dorsal root ganglia, liver and heart will be examined immunohistochemically to quantitate the amount of cellular frataxin and the reversal of iron accumulation and tissue degenerative changes. The activity of aconitases, as a measure of ISC-containing enzymatic function, will be also quantitated. An integrated efficacy score will be assigned to the tested compounds. A detailed pharmacokinetics/dynamics (ADME) analysis will eventually be started for those compounds which show the most promising activity.
  • ADME pharmacokinetics/dynamics
  • K 147 together with residues E 96 , E 100 , D 104 , F 127 , G 130 , L 103 , and A 99 surrounds a well defined cavity on the surface of frataxin (Fig. 4A-B). This cleft was chosen for in silico targeting in a virtual screening approach using the NCI chemical library.
  • the area near K 147 of frataxin, as defined by the x-ray structure of the protein (PDB code: 1EKG) was used as target. This site includes E100, L103, D104, F127, G128, SI 29, G130, and K 147 .
  • the area near K 147 as defined by NMR models for frataxin (PDB code 1LY7) was also used.
  • the available NMR structures (15 models) were analyzed with the same procedure used for the x-ray structure (program PASS). Three out of fifteen NMR models show a large pocket in the vicinity of K 147 .
  • model #7 in 1LY7 has been chosen for the docking studies, because it shows the largest pocket and because it is the most similar to the x-ray structure.
  • the virtual screening of the selected compounds has been carried out using the docking program Autodock/Vina.
  • the docking region has been centered on the coordinates of the Cb of K147, with a grid of 16x16x16 A with spacing of 0.375A.
  • the docked structures were sorted by affinity for 1EKG and 1LY7, and 61 were selected for further analysis.
  • Example selected compounds include:
  • Bacterial frataxin CyaY is the gatekeeper of iron-sulfur cluster formation catalyzed by IscS. Nat Struct Mol Biol 16, 390-396 (2009).
  • Saeki, Y., et al. Lysine 63-linked polyubiquitin chain may serve as a targeting signal for the 26S proteasome.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Epidemiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Neurosurgery (AREA)
  • Neurology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Hospice & Palliative Care (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Psychiatry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
  • Nitrogen- Or Sulfur-Containing Heterocyclic Ring Compounds With Rings Of Six Or More Members (AREA)
  • Plural Heterocyclic Compounds (AREA)

Abstract

L'invention concerne un procédé de traitement de l'ataxie de Friedreich avec des composés de formule I, notamment des sels, des tautomères ou des stéréo-isomères pharmaceutiquement acceptables des composés de formule Lp.
PCT/IB2010/003438 2009-12-07 2010-12-07 Compositions et procédés pour traiter l'ataxie de friedreich Ceased WO2011070444A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US13/514,288 US8703749B2 (en) 2009-12-07 2010-12-07 Compositions and methods for treating Friedreich's Ataxia

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US26734209P 2009-12-07 2009-12-07
US61/267,342 2009-12-07
US33214610P 2010-05-06 2010-05-06
US61/332,146 2010-05-06

Publications (2)

Publication Number Publication Date
WO2011070444A2 true WO2011070444A2 (fr) 2011-06-16
WO2011070444A3 WO2011070444A3 (fr) 2011-10-13

Family

ID=43857680

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2010/003438 Ceased WO2011070444A2 (fr) 2009-12-07 2010-12-07 Compositions et procédés pour traiter l'ataxie de friedreich

Country Status (2)

Country Link
US (1) US8703749B2 (fr)
WO (1) WO2011070444A2 (fr)

Families Citing this family (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
HUE028111T2 (en) * 2010-07-28 2016-11-28 Roberto Testi Frataxin mutants
WO2015017861A1 (fr) 2013-08-02 2015-02-05 Stealth Peptides International, Inc. Méthodes et compositions pour la prévention et le traitement de l'ataxie de friedreich
WO2015054662A1 (fr) 2013-10-10 2015-04-16 Eastern Virginia Medical School Dérivés de 4-((2-hydroxy-3-méthoxybenzyl)amino)benzènesulfonamide comme inhibiteur de la 12-lipoxygénase
KR101685993B1 (ko) * 2014-05-15 2016-12-13 한국과학기술연구원 전압개폐 칼슘이온채널 억제 활성화능을 갖는 ｎ-(피라졸릴메틸)아릴설폰아마이드 화합물 및 이를 포함하는 약학적 조성물
WO2016046759A2 (fr) * 2014-09-22 2016-03-31 Fratagene Therapeutics Ltd. Compositions et méthodes de traitement de l'ataxie de friedreich
US10442779B2 (en) * 2014-09-22 2019-10-15 Fratagene Therapeutics S.R.L. Compositions and methods for treating Friedreich's ataxia
WO2016103223A1 (fr) * 2014-12-23 2016-06-30 Fratagene Therapeutics Ltd. Procédés de traitement de l'ataxie de friedreich au moyen d'inhibiteurs de src
US10426775B2 (en) 2017-09-11 2019-10-01 Fratagene Therapeutics Srl Methods for treating Friedreich's ataxia with etravirine
AU2018338728B2 (en) 2017-09-29 2025-01-02 Centre National De La Recherche Scientifique (Cnrs) Rescue of central and peripheral neurological phenotype of Friedreich's Ataxia by intravenous delivery
EP3856762A1 (fr) 2018-09-28 2021-08-04 Voyager Therapeutics, Inc. Constructions d'expression de frataxine comprenant des promoteurs modifiés et leurs méthodes d'utilisation
JP7552614B2 (ja) * 2019-11-28 2024-09-18 三菱ケミカル株式会社 顕色剤及び感熱記録材料

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3322320A1 (de) * 1983-06-21 1985-01-03 Bayer Ag, 5090 Leverkusen Arylsulfonylhydrazone als treibmittel
JP2009530401A (ja) * 2006-03-24 2009-08-27 ザ・フェインスタイン・インスティチュート・フォー・メディカル・リサーチ フェノールヒドラゾンマクロファージ転移阻害因子阻害剤
SI22254A (sl) * 2006-04-13 2007-10-31 Univerza V Ljubljani Fakulteta Za Farmacijo NOVI ARILSULFONOHIDRAZIDNI INHIBITORJI ENCIMOV MurC IN MurD

Non-Patent Citations (40)

* Cited by examiner, † Cited by third party
Title
"Remington's Pharmaceutical Sciences", 1990, MACK PUBLISHING COMPANY
ACQUAVIVA, F. ET AL.: "Extra-mitochondrial localisation of frataxin and its association with IscU 1 during enterocyte-like differentiation of the human colon adenocarcinoma cell line Caco-2", J CELL SCI, vol. 118, 2005, pages 3917 - 3924
ADINOLFI, S. ET AL.: "Bacterial frataxin CyaY is the gatekeeper of iron-sulfur cluster formation catalyzed by IscS", NAT STRUCT MOL BIOL, vol. 16, 2009, pages 390 - 396
BERGE ET AL., J. PHARM. SCI., vol. 66, 1911, pages 1 - 19
BOUTET, S.C., DISATNIK, M.H., CHAN, L.S., LORI, K, RANDO, T.A: "Regulation of Pax3 by proteasomal degradation of monoubiquitinated protein in skeletal muscle progenitors", CELL, vol. 130, 2007, pages 349 - 362
BRADY, G.P., JR, STOUTEN, P.F: "Fast prediction and visualization of protein binding pockets with PASS", J COMPUT AIDED MOL DES, vol. 14, 2000, pages 383 - 401, XP002602506
BULTEAU, A.L. ET AL.: "Frataxin acts as an iron chaperone protein to modulate mitochondrial aconitase activity", SCIENCE, vol. 305, 2004, pages 242 - 245
CAREY, SUNDBERG: "Advanced Organic Chemistry", 2007, SPRINGER SCIENCE+BUSINESS MEDIA LLC
CONDO ET AL.: "Molecular control of the cytosolic aconitaselIRP switch by extramitochondrial frataxin", HUM MOL GENET DOI:10.1093/HMG/DDP592, 2010
CONDO, I. ET AL.: "In vivo maturation of human frataxin", HUM MOL GENET, vol. 16, 2007, pages 1534 - 1540, XP002669060
CONDO, I., VENTURA, N., MALISAN, F., TOMASSINI, B., TESTI, R.: "A pool of extramitochondrial frataxin that promotes cell survival", J BIOL CHEM, vol. 281, 2006, pages 16750 - 16756, XP055254892, DOI: doi:10.1074/jbc.M511960200
DELATYCKI, M.B.: "Evaluating the progression of Friedreich ataxia and its treatment", J NEUROL, vol. 256, no. 1, 2009, pages 36 - 41, XP009120601, DOI: doi:10.1007/s00415-009-1007-y
DESHAIES, R.J., JOAZEIRO, C.A: "RING domain E3 ubiquitin ligases", ANNU REV BIOCHEM, vol. 78, 2009, pages 399 - 434, XP055005600, DOI: doi:10.1146/annurev.biochem.78.101807.093809
DHE-PAGANON, S., SHIGETA, R., CHI, Y.I., RISTOW, M., SHOELSON, S.E: "Crystal structure of human frataxi", JBIOL CHEM, vol. 275, 2000, pages 30753 - 30756
GERMAIN, D: "Ubiquitin-dependent and -independent mitochondrial protein quality controls: implications in ageing and neurodegenerative diseases", MOL MICROBIOL, vol. 70, 2008, pages 1334 - 1341
GOTTESFEID, J.M.: "Small molecules affecting transcription in Friedreich ataxia", PHARMACOL THER, vol. 116, 2007, pages 236 - 248, XP022272070, DOI: doi:10.1016/j.pharmthera.2007.06.014
HABELHAH, H. ET AL.: "Regulation of 2-oxoglutarate (alpha-ketoglutarate) dehydrogenase stability by the RING finger ubiquitin ligase Siah", J BIOL CHEM, vol. 279, 2004, pages 53782 - 53788, XP002755670, DOI: doi:10.1074/jbc.M410315200
IRWIN, J.J, SHOICHET, B.K: "ZINC--a free database of commercially available compounds for virtual screening", J CHEM INFMODEL, vol. 45, 2005, pages 177 - 182, XP055113478, DOI: doi:10.1021/ci049714+
IWAI, K., TOKUNAGA, F: "Linear polyubiquitination: a new regulator of NF-kappaB activation", EMBO REP, vol. 10, 2009, pages 706 - 713
KOMANDER, D: "The emerging complexity of protein ubiquitination", BIOCHEM SOC TRANS, vol. 37, 2009, pages 937 - 953
KRAVTSOVA-IVANTSIV, Y., COHEN, S., CIECHANOVER, A: "Modification by single ubiquitin moieties rather than polyubiquitination is sufficient for proteasomal processing of the p105 NF-kappaB precursor", MOL CELL, vol. 33, 2009, pages 496 - 504
LI, W. ET AL.: "Genome-wide and functional annotation of human E3 ubiquitin ligases identifies MULAN, a mitochondrial E3 that regulates the organelle's dynamics and signaling", PLOS ONE, vol. 3, 2008, pages E1487
MARMOLINO, D, ACQUAVIVA, F: "Friedreich's Ataxia: from the (GAA)u repeat mediated silencing to new promising molecules for therapy", CEREBELLUM, vol. 8, 2009, pages 245 - 259, XP002669059, DOI: doi:10.1007/S12311-008-0084-2
MUSCO, G. ET AL.: "Towards a structural understanding of Friedreich's ataxia: the solution structure of frataxin", STRUCTURE, vol. 8, 2000, pages 695 - 707, XP008056061, DOI: doi:10.1016/S0969-2126(00)00158-1
PANDOLFO, M, PASTORE, A: "The pathogenesis of Friedreich ataxia and the structure and function of frataxin", JNEUROL, vol. 256, no. 1, 2009, pages 9 - 17, XP019714420
PANDOLFO, M.: "Friedreich ataxia: the clinical picture", JNEUROL, vol. 256, no. 1, 2009, pages 3 - 8, XP019714419
PUCCIO, H.: "Multicellular models of Friedreich ataxia", J NEUROL, vol. 256, no. 1, 2009, pages 18 - 24, XP019714421
ROTIN, D., KUMAR, S: "Physiological functions of the HECT family of ubiquitin ligases", NAT REV MOL CELL BIOL, vol. 10, 2009, pages 398 - 409
SAEKI, Y. ET AL.: "Lysine 63-linked polyubiquitin chain may serve as a targeting signal for the 26S proteasome", EMBO J, vol. 28, 2009, pages 359 - 37 1
SCHMUCKER, S., ARGENTINI, M., CARELLE-CALMELS, N., MARTELLI, A., PUCCIO, H: "The in vivo mitochondrial two-step maturation of human frataxin", HUM MOL GENET, vol. 17, 2008, pages 3521 - 3531, XP002669061
SCHULZ, J.B., DI PROSPERO, N.A, FISCHBECK, K: "Clinical experience with high-dose idebenone in Friedreich ataxia", JNEUROL, vol. 256, no. 1, 2009, pages 42 - 45, XP019714425
SCHWARTZ, A.L., CIECHANOVER, A: "Targeting proteins for destruction by the ubiquitin system: implications for human pathobiology", ANNU REV PHARMACOL TOXICOL, vol. 49, 2009, pages 73 - 96
TEAGUE, S.J., DAVIS, A.M., LEESON, P.D., OPREA, T: "The Design of Leadlike Combinatorial Libraries", ANGEW CHEM INT ED ENGL, vol. 38, 1999, pages 3743 - 3748
TREIER, M., STASZEWSKI, L.M., BOHMANN, D: "Ubiquitin-dependent c-Jun degradation in vivo is mediated by the delta domain", CELL, vol. 78, 1994, pages 787 - 798, XP024246455, DOI: doi:10.1016/S0092-8674(94)90502-9
TROTT, O., OLSON, A.J.: "AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading", J COMPUT CHEM, vol. 31, 2010, pages 455 - 461
TSOU, A.Y., FRIEDMAN, L.S., WILSON, R.B., LYNCH, D.R: "Pharmacotherapy for Friedreich ataxia", CNS DRUGS, vol. 23, 2009, pages 213 - 223
WRIGHT, G., TERADA, K., YANO, M., SERGEEV, I., MORI, M: "Oxidative stress inhibits the mitochondrial import of preproteins and leads to their degradation", EXP CELL RES, vol. 263, 2001, pages 107 - 117
XU, P. ET AL.: "Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation", CELL, vol. 137, 2009, pages 133 - 145, XP002689016, DOI: doi:10.1016/J.CELL.2009.01.041
YONASHIRO, R. ET AL.: "A novel mitochondrial ubiquitin ligase plays a critical role in mitochondrial dynamics", EMBO J, vol. 25, 2006, pages 3618 - 3626
YOON, T., COWAN, J.A: "Iron-sulfur cluster biosynthesis. Characterization of frataxin as an iron donor for assembly of [2Fe-2S] clusters in ISU-type proteins", J AM CHEM SOC, vol. 125, 2003, pages 6078 - 6084

Also Published As

Publication number Publication date
US20130109658A1 (en) 2013-05-02
WO2011070444A3 (fr) 2011-10-13
US8703749B2 (en) 2014-04-22

Similar Documents

Publication Publication Date Title
US8703749B2 (en) Compositions and methods for treating Friedreich's Ataxia
Skaar et al. Mechanisms and function of substrate recruitment by F-box proteins
Agola et al. A competitive nucleotide binding inhibitor: in vitro characterization of Rab7 GTPase inhibition
EP1554394B1 (fr) Méthode de screening utilisant un modéle structural de FIH
CA2904985C (fr) Compose de pyrazole-amide et utilisations medicinales s'y rapportant
US9586890B2 (en) Screening methods for the binding affinity of chemical entities to biological molecules and NEDD4-1 inhibitors identified by the screening methods
US20180256577A1 (en) KSR Antagonists
Burkard et al. Enabling and disabling polo-like kinase 1 inhibition through chemical genetics
Rufini et al. Highly specific ubiquitin-competing molecules effectively promote frataxin accumulation and partially rescue the aconitase defect in Friedreich ataxia cells
KR20150022911A (ko) B형 간염 바이러스 cccdna 전사의 조절
Bata et al. Inhibitors of the Hippo pathway kinases STK3/MST2 and STK4/MST1 have utility for the treatment of acute myeloid leukemia
US20110269141A1 (en) Target protein and target gene for drug discovery, and screening method
US9261497B2 (en) Method of treating cancer with modulators of SCFSkp2
US10889585B2 (en) Inhibitors of kidney-type glutaminase, GLS-1
Cirillo et al. Isoform-selective targeting of PI3K: time to consider new opportunities?
Brusa et al. Innovative strategy toward mutant CFTR rescue in cystic fibrosis: design and synthesis of thiadiazole inhibitors of the E3 ligase RNF5
US10273208B2 (en) Screening methods for the binding affinity of chemical entities to biological molecules and NEDD4-1 inhibitors identified by the screening methods
Bissegger et al. Rapid, potent, and persistent covalent chemical probes to deconvolute PI3Kα signaling
WO2016046759A2 (fr) Compositions et méthodes de traitement de l'ataxie de friedreich
Zhang et al. Identification of benzofuro [2, 3-b] pyridine pseudonatural product as a novel class of ferritinophagy inhibitors that potently suppress ferroptosis
Peters et al. Zoning in on Tankyrases: a brief review on the past, present and prospective studies
WO2007050673A2 (fr) Inhibiteurs des kinases dependantes des cyclines
US20180209956A1 (en) HTS Assay for Identifying Small Molecule Inhibitors of RAD52 and Uses of Identified Small Molecule Inhibitors for Treatment and Prevention of BRCA-Deficient Malignancies
US10442779B2 (en) Compositions and methods for treating Friedreich's ataxia
Saadah et al. Synthesis, molecular docking, and in vitro activity of a novel angiotensin-converting enzyme 2 inhibitor, LMS1007: a potential molecule in Covid-19 and cancer treatments

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 10814771

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 13514288

Country of ref document: US

122 Ep: pct application non-entry in european phase

Ref document number: 10814771

Country of ref document: EP

Kind code of ref document: A2